The autoimmune lymphoproliferative syndrome (ALPS) is a human disorder due to defective lymphocyte apoptosis resulting in abnormalities in lymphocyte homeostasis. This produces a combination of lymphadenopathy, autoimmunity and increased risk of lymphoma. The diagnostic criteria used to diagnose ALPS include a triad of findings: lymphadenopathy, increased circulating alpha-beta double negative T cells and defective in vitro Fas mediated lymphocyte apoptosis. The majority of patients with ALPS have been found to have a heterozygous mutation in the gene encoding Fas (CD95) while a small number of patients have been found to have mutations in Fas ligand or caspase 10. However, a sizeable number of ALPS patients do not have mutations in the genes noted about and are categorized as having ALPS type 3 with an uncharacterized defect. We have beegun a systematic evaluation of ALPS type 3 patients by sequentially studying intracellular proteins involved with the apoptotic process. These are targeted at assessing the assemblage of the Death Inducing Signalling Complex (DISC) and downstream events and proteins that control the apoptotic cascade that ultimately results in cell death. We have identified two ALPS type 3 patients who clearly fulfill the diagnostic triad for ALPS who have normal Fas, Fas ligand and caspase 8 by sequence analysis and assemble a normal DISC but still demonstrate defective Fas mediated lymphocyte apotosis in both activated T cells and an EBV transformed B cell line. In further studies, we have observed increased levels of an inhibitory protein, FLIP (Flice Inhibitory Protein) that is involved in the regulation of DISC mediated caspase activation. In particular, a specific isoform of FLIP, FLIP-s, is increased. We are currently studying the control mechanism(s) involved in FLIP regulation and attempting to replicate the defect seen in these patients by using siRNA knock down techniques in normal lymphocytes. We are also in the process of screening additional ALPS type 3 patients to determine the frequency of this abnormality among this subgroup of ALPS.